The mycobacteria are a genus of bacteria that are characterized as acid-fast, non-motile, gram-positive bacillus. The genus comprises many species including Mycobacterium africanum, M. avium, M. bovis, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. intracellulare, M. kansasii, M. leprae, M. microti, M. scrofulaceum, M. paratuberculosis, and M. tuberculosis. Some of the mycobacteria are pathogenic to both humans and animals, in particular M. tuberculosis, M. leprae, and M. bovis. Other mycobacterial species are not normally pathogenic, but cause opportunistic infections in immunocompromised individuals, such as AIDS patients. For example, infection by M. kansasii, M. avium, and M. intracellulare can cause severe lung disease in subjects in whom the immune system is suppressed or compromised. In fact, for the first time since 1953, reported cases of mycobacterial infections are increasing in the United States; many of these cases are related to the AIDS epidemic.
Detection of Mycobacterium species in clinical species is important as a clinical diagnostic tool. Historically, M. tuberculosis was thought to be the only clinically significant pathogen in this genus. A rise in the incidence of drug-resistant strains of M. tuberculosis has further emphasized the need to detect this species. Tuberculosis displays all of the principal characteristics of a global epidemic disease. Currently, tuberculosis afflicts more than 35 million individuals worldwide and results in over 4 million deaths annually. Thus, tuberculosis is a problem of major concern throughout the world. Tuberculosis can be caused by M. tuberculosis, M. bovis, M. africanum and M. microti, the acid-fast, Gram positive, tubercle bacilli of the family Mycobacteriaceae. Some local pathogenic strains of M. tuberculosis have also been isolated from patients in Madras and other cities in India, which differ in some respects from M. tuberculosis H37Rv, which is a virulent strain.
Other Mycobacterium species, however, are also clinically important. These are sometimes referred to as “MOTT” for mycobacterium other than tuberculosis, commonly including M. avium/intracellulare complex organisms (M. avium, M. intracellulare, M. paratuberculosis, commonly referred to as MAIC), M. gordonae, M. fortuitum, M. chelonae, M. mucogenicum and mixtures of Mycobacterium species in a clinical specimen. For example, fast-growing opportunistic infections by M. avium complex (MAC) bacteria have been shown to occur frequently in AIDS and other immunocompromised individuals. In such infected individuals, at least 106 MAC cells/ml of sputum sediment have been found. Therefore, detection assays that can detect many species of mycobacterium are clinically important.
Many clinical methods for detecting and identifying mycobacterium species in samples require analysis of the bacteria's physical characteristics (e.g., acid-fast staining and microscopic detection of bacilli), physiological characteristics (e.g., growth on defined media) or biochemical characteristics (e.g., membrane lipid composition). These methods require relatively high concentrations of bacteria in the sample to be detected, may be subjective depending on the clinical technician's experience and expertise, and are time-consuming. Because mycobacterium species are often difficult to grow in vitro and may take several weeks to reach a useful density in culture, these methods can also result in delayed patient treatment and costs associated with isolating an infected individual until the diagnosis is completed.
Mycobacteria in general, and M. tuberculosis and M. bovis in particular, are fastidious microorganisms which are very slow growing. It can take two to three weeks to grow these organisms on the culture media conventionally used. There have been several efforts to find a medium or a substance which can enhance the growth and reduce the time factor. For Example, U.S. Pat. No. 3,935,073, which is incorporated herein by reference, discloses a growth medium for culturing mycobacteria containing the following nutrients at the indicated levels: 7H9 broth base 0.47% (containing potassium and sodium phosphates, sodium glutamate, sodium citrate, ammonium sulfate, pyridoxine, ferric ammonium citrate, magnesium sulfate, zinc sulfate, copper sulfate, biotin and calcium chloride obtained from BBL Microbiology Systems in Cockeysville, Md.), bovine serum albumin 0.5%, casein hydrolysate 0.1%, catalase 96 units/vial, 14C labeled substrate 2 uCi/vial, deionized water balance to 2 ml, Final pH 6.8±0.1.
Albumin can be used as a detoxifying agent in a medium for the growth of mycobacteria. Albumin is a simple protein found in nearly every animal and in many vegetable tissues. Albumins are characterized as being soluble in water and coagulable by heat. They contain carbon, hydrogen, nitrogen, oxygen and sulfur. A preferred albumin for the growth media of the present invention is bovine serum albumin. The albumin is typically present in the growth media at a level of from about 0.1 percent by weight to about 10 percent by weight.
However, because of the slow growth rate and the need for an enriched medium for mycobacterial growth in culture, detection of mycobacteria from clinical samples (e.g. sputum, lung fluids, tissue or feces) still represents a significant biological challenge. One factor in this challenge arises from the fact that more rapidly-growing bacteria can overgrow the slow-growing mycobacterial organism of interest, thus precluding or significantly hindering mycobacteria detection. Over the decades, several techniques have been developed to decontaminate diagnostic samples (i.e. kill or inhibit non-mycobacterial organisms) submitted for mycobacterial identification. These techniques either kill the potential contaminates or injure them to the extent that their growth is inhibited or totally prevented.
Conventionally, laboratory diagnosis of mycobacteria was based on acid-fast staining and cultivation of the organism, followed by biochemical assays. As a result of the slow growth and long generation time of mycobacteria, accurate laboratory diagnosis of mycobacteria by conventional techniques can take as long as six weeks. Automated culturing systems such as the BacT/ALERT® system (bioMérieux, Inc.) can decrease the time for identification of mycobacteria by up to two weeks.
The present assignee, bioMérieux, Inc. offers the plastic BacT/ALERT® MP culture bottle for use in the BacT/ALERT® Microbial Detection systems as its culture based system for detecting mycobacteria in clinical samples, other than blood. The BacT/ALERT® Microbial Detection System utilizes a colorimetric sensor and reflected light to monitor the presence and production of carbon dioxide (CO2) that is dissolved in the culture medium. If mycobacteria are present in the test sample, carbon dioxide is produced as the microorganisms metabolize the substrates in the culture medium. When growth of the mycobacteria produces CO2, the color of the gas-permeable sensor installed in the bottom of each culture bottle changes from blue-green to yellow.
The BacT/ALERT® MP reagent system includes BacT/ALERT® MP culture bottle, reconstitution fluid (RF) and MB BacT antibiotic supplement (MAS) (hereinafter referred to as conventional or old RF (or conventional/old RF) and conventional or old MAS (or conventional/old MAS). The BacT/ALERT® MP culture bottle is a plastic bottle that contains some components of the medium. The reconstitution fluid (RF) contains the remaining nutrients for mycobacteria growth and is used to reconstitute the MAS. The MAS is a lyophilized powder made up of six antimicrobials to suppress unwanted respiratory flora from sputum samples. The RF and MAS are packaged as the MAS kit. Nevertheless, there still exists a need in the art to further reduce the time required for accurate diagnosis of mycobacteria.
It is, accordingly, a primary object of this invention to provide a culture media and method for enhanced growth and detection of Mycobacterium species that may be present in a clinical sample. A further object of this invention is to provide a novel mycobacteria culture medium suitable for the in vitro cultivation of mycobacteria. Still another object of this invention is to provide a mycobacteria culture medium in accordance with the preceding object, wherein growth of contaminating organisms is inhibited.
Accordingly, we describe herein a new culture media formulation, a new nutrient supplement (NS) and a new mycobacterial antimicrobial supplement (MAS) formulation, and manufacturing process improvements that show unexpected enhancement in the growth and detection of mycobacteria. Also described herein are methods for enhanced growth and detection of mycobacteria.